Tumor growth and metastasis depend upon angiogenesis that, in turn, requires growth factors, proteases, and other cofactors including copper. The following two lines of research suggest that copper could be used as a novel selective target for cancer therapies. (i) Copper, but not other transition metals, is a co-factor essential for tumor angiogenesis processes;high levels of copper have been found in many types of human cancers, including prostate, breast, colon, lung and brain. (ii) For human and mammals, there are no known harmful effects observed in normal organs (e.g., liver) when the bioavailability of copper is decreased by up to 80% below baseline. Our previous studies have suggested that CQ (clioquinol, a potent antibiotic that has come into recent interest for its potential use as a therapeutic agent for Alzheimer's Disease) may interact with prostate tumor cellular copper to form a proteasome inhibitor, resulting in down-regulation of androgen receptor (AR) protein expression and induction of apoptotic cell death in vitro and in vivo, which could account for the observed anti-tumor activity of CQ. However, the molecular basis for the in vivo mechanisms of CQ anti-tumor activity is still unclear. Does CQ affect the Cu accumulation, distribution and uptake by normal and tumor cells? Does CQ only target tumor cellular Cu with no effect on normal cells? It has been observed that CQ interact with Cu(II) in solution but this interaction might be expected to be different in vivo since most of the Cu available in vivo is Cu(I). The ways in which CQ affects the Cu chemical environment in vivo is unknown. An emerging technique that could be ideal, potentially, for answering such questions is micro-X-ray fluorescence spectroscopy. This a type of scanning probe microscopy where a 3-20 micron sized incident x-ray beam is used to excite transition metal atoms that are present in tissue sections and the resulting fluorescent x-rays used to measure both the quantity and the chemical state of the metal in question. The goal of this proposal is to evaluate a new detection system that has both sufficiently high energy resolution and high detection efficiency for the purpose of in vivo Copper speciation studies of biological systems with specific applications to cancer biology. It is proposed to determine high resolution spectra of Cu spectral lines of Cu model compounds and normal and tumor tissue sections using a high resolution analyzer to identify the spectral features of each compound. The model compounds have a well-known Cu oxidation state, coordination number and electronic geometrical configuration. The spectra will be used to generate a library of model spectra. Successful completion of the aims of this proposal will allow establishing the utility of micro-X-ray fluorescence spectroscopy for determining the roles of Cu speciation in anti-tumor studies. An understanding of the in vivo mechanism of Cu-ligands when acting as anti-tumor agents is an essential step towards new therapies that show reduced to very low toxicity to patients. The library of model spectra generated during this project will be an invaluable resource for other tissue studies involving Cu as a target. This project will open new frontiers for other biological studies that will require a high resolution analytical tool. PUBLIC HEALTH RELEVANCE: This proposal will evaluate a new detection system that has both sufficiently high energy resolution and high detection efficiency for the purpose of in vivo Copper speciation studies of biological systems with specific applications to cancer biology. Successful completion of this proposal will allow establishing the utility of micro-X-ray fluorescence spectroscopy for determining the roles of Cu speciation in anti-tumor studies. An understanding of the in vivo mechanism of Cu-ligands when acting as anti-tumor agents is an essential step towards new therapies that show reduced to very low toxicity to patients. This project will open new frontiers for other biological studies that will require a high resolution analytical tool. 1